Note: Descriptions are shown in the official language in which they were submitted.
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Antioxidant composition
FIELD OF THE INVENTION
The present invention relates to compositions comprising an edible oil having
improved
oxidative stability and methods for manufacturing such compositions.
BACKGROUND OF THE INVENTION
Omega-3 fatty acids are considered essential fatty acids, which means that
they are
essential to human health but cannot be synthesised by the human body. For
this reason,
omega-3 fatty acids must be obtained through the diet.
The European Pharmacopoeia defines the omega-3 fatty acids as the following
acids
(see for example Monograph no. 1912, Fish Oil, Rich in Omega-3-Acids): alpha-
linolenic acid (018:3 n-3; ALA), moroctic acid (C18:4 n-3), eicosatetraenoic
acid,
(C20:4 n-3), eicosapentaenoic (timnodonic) acid (C20:5 n-3; EPA),
heneicosapentaenoic acid (C21:5 n-3), docosapentaenoic (clupanodonic) acid
(C22:5 n-
3) and docosahexaenoic (cervonic) acid (C22:6 n-3; EPA). Omega-3 fatty acids
with
chain-length of 20 and above are called long-chain omega-3 fatty acids. ALA is
common in a number of vegetable oils. C 18:4 n-3 is available from fish oils,
as well as
from some vegetable oils. Once eaten, the body can to some extent convert ALA
and
C 18:4 n-3 to the long-chain omega-3 fatty acids, including EPA and DHA.
However,
fish oil and other marine oils are known to be the best source of these omega-
3 fatty
acids. Long-chain omega-3 fatty acids can also be obtained via fermentation of
single
cell oils (microbial oils), and research projects aim at producing EPA and DHA
via
gene-modified terrestrial plants.
Omega-3 fatty acids have been demonstrated to reduce the risk of coronary
heartõ
disease as well as having a positive effect on children's development, as well
as on the
skin. Results have also been disclosed indicating the positive effect of these
fatty acids
on certain mental illnesses, autoimmune diseases and joint complaints. There
are
therefore many reasons for considering taking fish oil as a valuable dietary
supplement,
including the long-term effect which this dietary supplement is now thought to
have.
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However, fish oils and especially concentrates of omega-3 fatty acids are-
very,.
susceptible to oxidation. Oxidation limits the use of such products in food
applications,
and also limits oral administration of omega-3 containing nutritional
supplements,
except where the supplements are encapsulated.
Microencapsulation is a way of formulating omega-3 oils for food applications.
However, microencapsulated products are relatively expensive, the
encapsulation
material often takes up more volume than the oil, so that the total volume
becomes
impractical to handle, and also there might be doubts whether the
encapsulation material
io prevents the valuable omega-3 fatty acids from being absorbed in the
intestinal tract.
An alternative to microencapsulation is the use of antioxidants in the oil to
slow down
oxidation. Most antioxidants interfere with the propagation of lipid oxidation
by
donating a hydrogen atom to, and thereby inactivating, chain-carrying peroxyl,
radicals
and/or alkoxyl radicals. Hence, after breaking the chain reaction of lipid
peroxidation,
an antioxidant is itself converted to a radical. To be effective, the
antioxidant radical
has to be sufficiently stable so as to react slowly with the lipid substrate
and rapidly
with lipid peroxyl radicals and/or alkoxyl radicals. Several natural and
synthetic
compounds fulfil this condition and are widely used for preserving
polyunsaturated fatty
acids (PUFA) from oxidative deterioration. Even though a number of synthetic
antioxidants have been extensively used for the stabilization of foods, much
interest has
developed in the use of naturally occurring antioxidants because of the
adverse attention
received by the synthetic antioxidants and because of the worldwide trend to
avoid or
minimize the use of synthetic food additives.
Tocopherols are among the most important lipid-soluble natural antioxidants,
and
appear to be the major physiological scavengers of free radicals inside human.
membranes and plasma lipids. The fact that these compounds are naturally
occurring
lipid-soluble antioxidants make them particularly useful in combination. with
marine.
oils, having high amounts of PUFA, intended for human consumption (Free
radical.,.
biology & medicine, 2005, vol. 38, page 78-84; J. Chem. Soc., Perkin Trans.
.2; 1998).
Another example of antioxidants commonly used in combination with marine oils
are
rosemary extracts. The antioxidant potential of such an extract has previously
been
tested on cod liver oil and has been shown to have significantly higher
antioxidant
effect compared with seven other naturally occurring antioxidants (Journal of
Aquatic
Food Product Technology; vol. 14; 2005; page 75-94). Further, a mixture of a-
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tocopherol and rosemary extract has previously been shown to exert very strong
antioxidant activity in sardine oil, where their combination not only
inhibited the
formation of hydroperoxides much more effectively than when present separately
but
the activity of tocopherol was retained for a longer period of time (Yukagaku
1994, vol. 43, no2, page 109-115).
Ascorbyl palmitate is an ester formed from ascorbic acid and palmitic acid. In
addition
to its use as a source of vitamin C, it is also commonly used as an
antioxidant food
additive. The compound is difficult to dissolve in oil formulations, and it is
therefore
common to add lecithin to the antioxidant preparation in order to solubilise
the ascorbyl
palmitate (EP612346). Even though lecithin is commonly regarded as a well-
tolerated
and non-toxic surfactant, lecithin may contain traces of proteins and for this
reason has
to be declared as a potential allergen in nutritional supplements. Producers
and
distributors prefer to avoid components that might act as allergens.
In addition to traditional and cultural reasons for consuming tea, a renewed
interest has
been fuelled by the discovery of strong antioxidant properties provided by tea
prepared
from Camellia Sinensis leaves (green tea). Such an antioxidant effect has
primarily been
attributed to the polyphenol content of the tea leaves, commonly known as tea
catechins. Said catechins are water-soluble and therefore not easily
dissolvable in'oil
formulations. In order to make these strong antioxidants lipid-soluble, it has
been
suggested to derivatise part of the phenols with fatty acid (WO07021789) An.
unwanted side effect of derivatising these compounds with fatty acids is -that
the
intestinal absorption of these compounds increases significantly. Polyphenols
from
green tea are generally not absorbed into the body, and the increased
absorption of these
compounds add a problem from a regulatory point of view.
Even though a number of antioxidants and various combinations thereof have
been
disclosed (e.g. US5102659), there is still a need for additional antioxidant
compositions
having improved characteristics.
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SUMMARY OF THE INVENTION
It has previously been disclosed that green tea extracts have strong
antioxidant activity.
However, most of the compounds responsible for said antioxidant activity
arewater-
s soluble and therefore not easily dissolvable in oil formulations. It has
been suggested. to
derivatise the active compounds with fatty acids, but since this may add a
problem from
a regulatory point of view, it is desirable to avoid this strategy.
Accordingly, it is an object of the present invention to provide a composition
io comprising an edible oil, preferably an edible marine oil, and green tea
extract without
having to chemically modify the green tea catechines.
Surprisingly, the inventors of the present invention have observed that the
solubility of
the green tea catechines is highly dependent on the content of partial
glycerides in the
15 composition.
Thus, a first aspect of the present invention relates to a composition
comprising an
edible oil and green tea extract or an active fraction of said green tea
extract, said
composition containing more than 2 % partial glycerides by weight of the
composition.
As previously disclosed, partial glycerides have been shown to significantly
increase the
solubility of green tea catechines in oily formulations. Of particular
interest are citric
acid and tartaric acid esters of partial glycerides which have been shown
especially
suitable for dissolving green tea catechines in oily formulations.
Thus, a third aspect of the present invention relates to composition
comprising an edible
oil, green tea extract or an active fraction of said green tea extract and
component A,
wherein component A is a compound of formula (I) or a salt thereof; or a
mixture of
different compounds each represented by formula (I) or a salt thereof,
0 0 0
W N -~
Formula (I)
wherein o
Rl represents --0(CH2)õ CH3;
n is an integer in the range 3-30;
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R2 and R3 are independently selected from the group consisting of
p COOH
o R5 0
CH2CCH2COOH IL i COOK , --0-(CH2),,CH3 and -H;
11 R4 5 'OH
in is an integer in the range 3-30;
0
R4 and R5 are independently selected from OHS c-to- and -OH;
0
io with the proviso that at least one of R2 and R3 is other than -C(CH2)mCH3
and -H; and
that at least one of R4 and R5 is -OH.
A second aspect of the present invention relates to a method for manufacturing
the
composition according to present invention, the method comprising the
following steps:
a) optionally, green tea extract or an active fraction thereof is mixed with
ascorbyl
palmitate;
b) an edible oil is mixed with partial glycerides so that the final
composition contains
more than 2 % partial glycerides by weight of the final composition (if said
edible
oil contains more than 2 % partial glycerides by weight of the final
composition, it
is not necessary to add further partial glycerides); or an edible oil is mixed
with
component A, as defined in the third aspect of the present invention;
c) green tea extract or an active fraction thereof or the product obtained in
step a) is
mixed with the product obtained in step b);
d) the product obtained in step c) is subjected to filtration;
e) optionally, at least one tocopherol or tocopherol derivative and/or
rosemary extract
or an active fraction thereof is added to the product obtained in step a), b),
c) and/or
d).
A fourth aspect of the present invention relates to a method for manufacturing
the
composition according to the present invention, the method comprising the
following
steps:
a) green tea extractor an active fraction thereof is mixed with a lower
alcohol (C1_6
alcohol), such as ethanol;
b) the product obtained in step a) is subjected to filtration;
c) optionally, the filtrate obtained in step b) is mixed with ascorbyl
palmitate;
d) component A, as defined in the third aspect of the present invention, or
partial
glycerides, as defined in the first aspect of the present invention is added
to the
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product obtained in step b) or step c); or optionally directly to the edible
oil defined
in step g);
e) optionally, at least one tocopherol or tocopherol derivative and/or
rosemary extract
or an active fraction thereof is added to the product obtained in step a), b),
c) or.d);
s f) the lower alcohol is removed; and
g) the product obtained in step f) is mixed with an edible oil.
A fifth aspect of the present invention relates to a nutritional, food or
pharmaceutical
composition, comprising the composition according to the first or third
aspect.of the
io present invention or the composition obtainable by the method according .1o
the second
or fourth aspect of the present invention.
DESCRIPTION OF THE FIGURES
is Figure 1 shows a synergistic antioxidative effect (induction time analysis)
from
combining a mixture of tocopherols (Tocoblend L 70 IP, Vitablend (1,03mg per
gram
marine oil)), rosemary extract (Oleoresin Rosemary, Kalsec (2,87mg per gram
marine
oil)) and ascorbyl palmitate (Grindox ascorbyl palmitate, Danisco (0,72mg per
gram
marine oil)) and olive oil (0,78mg olive oil per gram marine oil) with green
tea extract
20 (GUARDIANTM Green Tea Extract 20M , Danisco (5.4 mg per gram marine oil
before
filtration).
I represents Ascorbyl Palmitate+Tocopherols+Rosemary extract;
2 represents Green Tea Extract;
25 3 represents 1+2, "filtration, 20 C";
4 represents 1+2, "filtration, 80 C";
Figure 2 shows a synergistic antioxidative effect (increased weight analysis)
from,,
combining a mixture of tocopherols (Tocoblend L 70 IP, Vitablend (1,03mg per
gram
30 marine oil)), rosemary extract (Oleoresin Rosemary, Kalsec (2,87mg per gram
marine
oil)) and ascorbyl palmitate (Grindox ascorbyl palmitate, Danisco (0,72mg per
gram
marine oil)) and olive oil (0,78mg olive oil per gram marine oil) with green
tea extract
(GUARDIANTM Green Tea Extract 20M, Danisco (5.4 mg per gram marine oil before
filtration).
1 represents 2+Green Tea Extract, "filtration, 80'C",-
2 represents 4+Ascorbyl Palmitate+Tocopherols+Rosemary extract;
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3 represents 2+Green Tea Extract, "filtration, 20'C",-
4 represents EPAX 6000 EE;
Figure 3 shows the amount of green tea catechines dissolved in water and four
different
oil preparations. Dissolution in water gives a straight line that can be
utilised as a
calibration curve for estimation of the dissolved amount in the omega-3
containing oils.
Acid value number that expresses in milligrams the additional quantity of
potassium
hydroxide compared to the composition without green tea extract
required to neutralise 1 gram of the composition.
Mg/g active amount of catechins per gram oil
Water Water + guardian 20M (green tea extract)
EEl marine oil (5.6 % triglycerides (TG); 3.0 % diglycerides (DG); 3.6%
monoglycerides (MG); 87.8 % Ethyl esters/Free fatty acids (EE/FFA)) +
guardian 20M (green tea extract)
EE2 marine oil (0 % triglycerides (TG); 0.5 % diglycerides (DG); 12.0 %
monoglycerides (MG); 87.5 % Ethyl esters/Free fatty. acids (EE/FFA)) +
guardian 20M (green tea extract)
TGI marine oil (93.6 % triglycerides (TG); 4.9 % diglycerides (DG); 0.4 %
monoglycerides (MG); 0.5 % Ethyl esters/Free fatty acids (EE/FFA)) +
guardian 20M (green tea extract)
TG2 marine oil (63.9 % triglycerides (TG); 31.6 % diglycerides (DG); 1.9 %
monoglycerides (MG); 2.5 % Ethyl esters/Free fatty acids (EE/FFA))+
guardian 20M (green tea extract)
Figure 4 shows the amount of green tea catechines dissolved in water and a
marine oil
mainly containing ethyl esters and free fatty acids) with or without citrem.
Dissolution
in water gives a straight line that can be utilised as a calibration curve for
estimation of
the dissolved amount in the marine oil.
Acid value number that expresses in milligrams the additional quantity of
potassium
hydroxide compared to the composition without green tea' extract
required to neutralise 1 gram of the composition.
Mg/g active amount of catechins per gram oil
Water Water + guardian 20M (green tea extract)
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Figure 5 shows the amount of green tea catechines dissolved in a marine oil (
mainly
containing triglycerides) with or without citrem.
Acid value number that expresses in milligrams the additional quantity of
potassium
hydroxide compared to the composition without green tea extract
required to neutralise I gram of the composition.
Mg/g active amount of catechins per gram oil
Figure 6 shows the amount of green tea catechines dissolved in a marine oil
(mainly
containing ethyl esters and free fatty acids) with or without 2.1 %
monoglycerides by
weight of the final composition.
Acid value number that expresses in milligrams the additional quantity
ofpotassium
hydroxide compared to the composition without green tea extract
required to neutralise I gram of the composition.
Mg/g active amount of catechins per gram oil
DETAILED DESCRIPTION OF THE INVENTION
As previously disclosed, there are many reasons for considering taking fish
oil as a
valuable dietary supplement, including the long-term effect which this dietary
is supplement is now thought to have. However, fish oils and especially
concentrates of
omega-3 fatty acids are very susceptible to oxidation. Accordingly, there is a
need in
the art to provide an edible oil composition, particularly an edible marine
oil
composition, having improved oxidative stability.
It has previously been disclosed that green tea extracts contain compounds
which have
strong antioxidant activity. However, most of the compounds responsible for
said,:
antioxidant activity are water-soluble and therefore not easily dissolvable
in'oil
formulations. It has been suggested to derivatise the active compounds with
fatty acids
(WO07021789), but since this may add a problem from a regulatory point of
view, it is
desirable to avoid this strategy.
Accordingly, it is an object of the present invention to provide a composition
comprising an edible oil, preferably an edible marine oil, and green tea
extract without
having to chemically modify the green tea catechines.
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The antioxidant effect of green tea has primarily been attributed to the
polyphenol--
content of the tea leaves, commonly known as tea catechines. Based on the
assumption
that the active components of green tea extract to a large extent are present
as'
compounds like phenols, the dissolved amount may be analysed by titration with
potassium hydroxide. This approach has been used in order to investigate
a)whether the
amount of monoglycerides, diglycerides and triglycerides in the composition
may affect
dissolution of the green tea components (example 7, figure 3, figure 6); and
b)whether
there are any specific compounds that are particularly useful when it comes to
io increasing the amount of green tea components that may be dissolved in oil
formulations (example 8, example 10, figure 4 and figure 5).
The results of the experiments disclosed in example 7 clearly indicate that
when green
tea extract on maltodextrin carrier is added according to the present
invention to an
is ethyl ester or triglyceride oil containing partial glycerides, the green
tea components
that can be analysed by titration with potassium hydroxide can be dissolved
nearly
quantitatively in the oil. For a triglyceride with very low amounts of partial
glycerides
the dissolution is much lower. This positive dissolution promoting effect of
the partial
glycerides is very surprising.
Further, the results of the experiments disclosed in example 11 clearly
indicate that
when green tea extract on maltodextrin carrier is mixed with an ethyl ester to
which
monoglycerides have been added, the amount of green tea components that can be
analysed by titration with potassium hydroxide is significantly higher as
compared to
control (ethyl ester without added monoglycerides).
Thus, a first aspect of the present invention relates to a composition
comprising an,
edible oil and green tea extract or an active fraction of said green tea
extract, said.-
composition containing more than 2 % partial glycerides by weight of the
composition.
In one embodiment according to the present invention, the composition contains
mare
than 2.5 % partial glycerides by weight of the composition, preferably at
least 3'%
partial glycerides by weight of the composition (e.g. at least 4 % by weight
of the
composition), more preferably at least 5 % partial glycerides by weight of
the:.
composition (e.g. at least 6 % or 7 % by weight of the composition), even more
preferably at least 8 % partial glycerides by weight of the composition (e.g.
at least 9 %
by weight of the composition) and most preferably at least 10 % partial
glycerides by
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weight of the composition (e.g. at least 15 %, 17 %, 19 %, 21 %, 23 % or 25 %
by
weight of the composition).
Partial glycerides are esters of glycerol with fatty acids, where not all the
hydroxyl
5 groups are esterified (mono- and/or diglycerides).
In one embodiment according to the present invention, said partial
glycerides.' are
monoglycerides.
10 Accordingly, the present invention relates to a composition comprising an
edible oil and
green tea extract or an active fraction of said green tea extract, said
composition
containing more than 2 % monoglycerides by weight of the composition.
In one embodiment according to the present invention, the composition contains
more
is than 2.5 % monoglycerides by weight of the composition, preferably at least
3 %
monoglycerides by weight of the composition (e.g. at least 4 % by weight of
the
composition), more preferably at least 5 % monoglycerides by weight of the
composition (e.g. at least 6 % or 7 % by weight of the composition), even more
preferably at least 8 % monoglycerides by weight of the composition (e.g.
at.least 9 %
by weight of the composition) and most preferably at least 10 % monoglycerides
by
weight of the composition (e.g. at least 15 %, 17 %, 19 %, 21 %, 23 % or 25 %
o by
weight of the composition).
In another embodiment, said partial glycerides are diglycerides.
Accordingly, the present invention relates to a composition comprising an
edible oil and
green tea extract or an active fraction of said green tea extract, said
composition
containing more than 2 % diglycerides by weight of the composition.
In one embodiment according to the present invention, the composition contains
more
than 2.5 % diglycerides by weight of the composition, preferably at least 3 %
diglycerides by weight of the composition (e.g. at least 4 % by weight of the
composition), more preferably at least 5 % diglycerides by weight of the
composition
(e.g. at least 6 % or 7 % by weight of the composition), even more preferably.
at least '8
% diglycerides by weight of the composition (e.g. at least 9 % by weight of
the
composition) and most preferably at least 10 % diglycerides by weight of the
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composition (e.g. at least 15 %, 17 %, 19 %, 21 %, 23 % or 25 % by weight of
the
composition).
In yet another embodiment, said partial glycerides are a mixture of mono- and
diglycerides.
Accordingly, the present invention relates to a composition comprising an
edible oil and
green tea extract or an active fraction of said green tea extract, said
composition
containing more than 2 % of a mixture of mono- and diglycerides by weight of
the
io composition.
In one embodiment according to the present invention, the composition contains
more
than 2.5 % of a mixture of mono- and diglycerides by weight of the
composition,'
preferably at least 3 % of a mixture of mono- and diglycerides by weight of
the
composition (e.g. at least 4 % by weight of the composition), more preferably
at least 5
% of a mixture of mono- and diglycerides by weight of the composition (e.g. at
least 6
% or 7 % by weight of the composition), even more preferably at least 8 % of a
mixture
of mono- and diglycerides by weight of the composition (e.g. at least 9 % by
weight of
the composition) and most preferably at least 10 % of a mixture of mono- and
diglycerides by weight of the composition (e.g. at least 15 %, 17 %, 19 %, 21
%, 23 %
or 25 % by weight of the composition).
Said partial glycerides are preferably glycerol mono- and/or diesters of
unsaturated fatty
acids, preferably glycerol mono- and/or diesters of polyunsaturated fatty
acids-and most
preferably glycerol mono- and/or diesters of omega-3 fatty acids. Said fatty
acids .. `
preferably having 3-30 carbon atoms, more preferably 5-30 carbon atoms and
most.
preferably 10-30 carbon atoms.
Polyunsaturated fatty acids are fatty acids having 2 or more double bonds,
preferably
separated from each other by a single methylene group. Preferably, said
polyunsaturated
fatty acids have more than 2 double bonds, e.g. 3, 4, 5 or 6 double bonds that
preferably
are separated from each other by a single methylene group.
Omega-3 fatty acids are a family of unsaturated fatty acids that have in
common a final
carbon-carbon double bond in the n-3 position; that is, the third bond from
the methyl
end of the fatty acid. Nutritionally important n-3 fatty acids include a-
linolenic acid
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(ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), all of
which
are polyunsaturated.
By the expression partial glycerides by weight of the composition or by weight
of the
edible oil, there is meant the results from analysis by size-exclusion
chromatography
according to methods similar to those described in Ph.Eur. monographs 1352
(Omega-
3-acid triglycerides) and 2063 (Omega-3-acid ethyl esters 60).
By the expression omega-3 fatty acids by weight of the composition or by
weight of the
edible oil, there is meant not only the free omega-3 fatty acids but also
omega-3 fatty
acids in the form of e.g. glycerides (mono-, di- and triglycerides) and/or
ethyl ester.
For quantitative determination of the EPA and DHA content in omega-3
containing.
products and calculation of percentage content of the total omega-3 acids, see
'Ph.Eur.
2.4.29.
Further, the results of the experiments presented in example 8 and 10 clearly
indicate
that when green tea extract on maltodextrin carrier is added to an ethyl ester
or
triglyceride oil to which citric acid esters of partial glycerides have been
added, the
amount of green tea components present in the oil that can be analysed by
titration with
potassium hydroxide is significantly increased. This positive dissolution
promoting
effect of the citric acid esters of partial glycerides is very surprising.
Thus, a third aspect of the present invention relates to a composition
comprising an
edible oil, green tea extract or an active fraction of said green tea extract
and component
A, wherein component A is a compound of formula (I) or a salt thereof; or a
mixtur6 of
different compounds each represented by formula (I) or a salt thereof,
W N ~
Formula (I)
wherein o
Rl represents -C-(CH2)õCH3;
n is an integer in the range 3-30;
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R2 and R3 are independently selected from the group consisting of
0 COOH
0 R5
CH2CCH2COOH COON , --(CH2)mCH3 and H;:.
OH R4
m is an integer in the range 3-30;
0
11
R4 and R5 are independently selected from cH - c
--o- and -OH;
0
11
with the proviso that at least one of R2 and R3 is other than -- (CH2),,,CH3
and -H; and
that at least one of R4 and R5 is -OH.
As component A contains a plurality of acid groups, component A may exist in
i) non-
neutralized form; ii) partly neutralized form; iii) essentially neutralized
form or iv)
completely neutralized form, mainly depending on the pH of the solution in
which 'it is
dissolved.
In one embodiment according to the present invention, component A is in non-
neutralized form, more preferably in partly neutralized form and most
preferably in
essentially or completely neutralized form. Preferably said component A is
partly,
essentially or completely neutralized using sodium hydroxide or potassium
hydroxide.
0
The compound defined as -~0-(CH2)õCH3 or -,d11
-(CH2)mCH3 may be linear or branched,
preferably linear.
It is preferred that n is an integer in the range 5-30, such as 7-30, 9-30, 11-
30, 13-30,
15-30 or 16-30; more preferably n is an integer in the range 5-25, such as 7-
25, 9-25,
11-25, 13-25, 15-25 or 16-25; even more preferably n is an integer in the
range 5-20,
such as 7-20, 9-20, 11-20, 13-20, 15-20 or 16-20; and most preferably n is an
integer in
the range 5-16, such as 7-16, 9-16, 11-16, 13-16, 15-16, such as e.g. 16.
Further, it is preferred that m is an integer in the range 5-30, such as 7-
30;'9-30, 11;-30,
13-30, 15-30 or 16-30; more preferably m is an integer in the range 5-25, such
as 7-25,
9-25, 11-25, 13-25, 15-25 or 16-25; even more preferably m is an integer in
the range 5-
20, such as 7-20, 9-20, 11-20, 13-20, 15-20 or 16-20; and most preferably m is
an
integer in the range 5-16, such as 7-16, 9-16, 11-16, 13-16, 15-16, such as
e.g. 16.
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In one preferred embodiment m is equal to n.
In one preferred embodiment, R2 and R3 is independently selected from the
group
consisting of.
s
O COOH 0
~I
CH2C. 2COOH , - C-(CH2)mCH3 and -H;
OH 0
11
with the proviso that at least one of R2 and R3 is other than ---(CH2)mCH3 and
-H.
to
In another embodiment, R2 and R3 is independently selected from the group
consisting of-
0 R5 0
11 1 11
-CO H , - -(CH2),,,CH3 and -H;
15 R4 a
wherein R4 and R5 are independently selected from CH - --0- and -OH;
0
with the proviso that at least one of R2 and R3 is other than -04CH2),CH3 and -
H.,-,
0
20 In one preferred embodiment, R2 is -H or --g-(CH2)mCH3 and R3 is selected
from the
group consisting of-
0
7:--~ COOH
0
R5
CH2CC 2CCOH and --~----I-"_'COOH
25 OH R4 0
11
wherein R4 and R5 are independently selected from Cl C-0- and -OH;
0
In one preferred embodiment, R2 is -H or -a.-(CH2)mCH3 and R3 is
OOH
CH2CCH2COOH
OH
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O
In one preferred embodiment, R2 is -H or - (CH2),,,CH3 and R3 is
o R5
-dl Ii .coot
R4
5 0
11
wherein R4 and R5 are independently selected from CHI--b
-0- and -OH.
In one embodiment according to the present invention, component A is a mixture
of
different compounds each represented by formula (I) or a salt thereof.
to In one embodiment according to the present invention, component A
represents a
compound of formula (II) or a salt thereof; a compound of formula (III) or a
salt thereof;
or any mixture thereof
0
-OH
0 R5
0n 0 COOH
R4
Formula (II) Formula (III)
0
11
wherein R4 and R5 are independently selected from CH --0-o- and -OH.
In another embodiment, both R4 and R5 are -OH. In another embodiment R4 is -
OH. and
0
R5 is cH G--0- . In another embodiment, R4 is OHS--c-0- and R5 is -OH.
In one embodiment according to the present invention, component A represents a
compound of formula (II) or a salt thereof. In another embodiment, component A
represents a compound of formula (III) or a salt thereof. In another
embodiment,
component A represents a mixture of compounds each represented by formula II
or salts
thereof; or formula III or salts thereof.
In one embodiment according to the present invention the composition contains
at least
0.3 % component A by weight of the composition, preferably at least 0.35 %
component
A by weight of the composition, more preferably at least 0.4 % component A by
weight
of the composition, even more preferably at least 0.5 % component A by weight
of the
composition and most preferably at least 0.55 % component A by weight of the
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16
composition such as 0.6 %, 0.65 %, 0.7 %, 0.75 %, 0.8 %, 0.85 %, 0.9 %, 0.95
%, 1 %,
1.5%,2%,2.5%,3%,3.5%,4%, 4.5%,5 %, 5.5 % or 6 % by weight of the
composition.
In one embodiment according to the present invention, said composition does
not
contain a phenolic diterpene.
Diterpenes are natural substances having 20 carbon atoms which are made up of
four
isoprene units. They belong to the terpene group. Compounds designated
diterpenes are
io not only hydrocarbons, but also derivatives of the corresponding
hydrocarbons. .
Phenolic diterpenes are those diterpenes which contain at least one phenolic
OH group,
e.g. Carnosolic acid.
In another embodiment according to the present invention, the only source of
is triglycerides in said composition (particularly triglycerides of which each
fatty acid
have 6-18 carbon atoms, such as e.g. triglycerides the fatty acids of which
have
predominantly 8 or 10 carbon atoms) being the triglycerides that may be
present in the
edible oil.
20 In another embodiment according to the present invention, said composition
does not
contain triglycerides, particularly triglycerides of which each fatty acid
have 6-18
carbon atoms, such as e.g. triglycerides of which each fatty acid have
predominantly 8
or 10 carbon atoms. One example of a commercially available triglyceride
containing
composition, of which each fatty acid has predominantly 8 or 10 carbon atoms,
is
25 Delios V.
In one embodiment according to the present invention, said composition
contains less
than 3 % of a hydrophobic emulsifier by weight of the composition, e.g. less
than 2.5 %
of a hydrophobic emulsifier by weight of the composition, less than 2 % of a
30 hydrophobic emulsifier by weight of the composition, less than 1.5 % of a
hydrophobic
emulsifier by weight of the composition, less than 1 % of a hydrophobic
emulsifier by
weight of the composition or less than 0.5 % of a hydrophobic emulsifier by
weight of
the composition.
35 In another embodiment according to the present invention, a hydrophobic
emulsifier is
not added to the composition.
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17
A hydrophobic emulsifier has an HLB value of less than S. For determination of
HLB
values see: Tensid-Taschenbuch [surfactant Handbook], 2"d edition, edited by
H.
Stache, Carl Hanser Verlag, 1981.
In one embodiment, said hydrophobic emulsifier is selected from the group
consisting
of a polyglycerol polyricinolate, a polyglycerol ester of fatty acid and
glycerol
monoester of fatty acid (e.g. glycerol monooleate and glycerol monostearate).
In another
embodiment said hydrophobic emulsifier is mono- and/or diesters of mono-
unsaturated
fatty acids and/or mono- and/or diesters of unsaturated fatty acids.
In one embodiment according to the present invention, said composition
contains less
than 3 % water by weight of the composition, e.g. less than 2.5 % water by
weight of
the composition, less than 2 % water by weight of the composition, less than
1.5 %:
water by weight of the composition, less than 1 % water by weight of the
composition,
less than 0.5 % water by weight of the composition, less than 0.05 % water by
weight of
the composition, less than 0.01 % water by weight of the composition or less
than 0.005
% water by weight of the composition.
In another embodiment according to the present invention, the composition is
essentially or totally free of water.
By the expression "edible oil" there is meant an edible oil or an oil derived
therefrom.
Said edible oil may be of animal or vegetable origin. Examples of oil derived
from an
edible oil are ethyl esters and free fatty acids.
In one embodiment the edible oil is in the form of triglycerides,
diglycerides,
monoglycerdies, ethyl esters, free fatty acids or any combination thereof.
Said partial glycerdies are preferably glycerol mono- and/or diesters of
unsaturated fatty
acids, preferably glycerol mono- and/or diesters of polyunsaturated fatty
acids and most
preferably glycerol mono- and/or diesters of omega-3 fatty acids. Said fatty
acids
preferably having 3-30 carbon atoms, more preferably 5-30 carbon atoms
and=most
preferably 10-30 carbon atoms.
In one preferred embodiment, said edible oil is in the form of ethyl esters,
preferably
ethyl esters of unsaturated fatty acids (e.g. of long-chain unsaturated fatty
acids) more
preferably ethyl esters of polyunsaturated fatty acids (e.g. of long-chain
polyunsaturated
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18
fatty acids) most preferably ethyl esters of omega-3 fatty acids (e.g. of long-
chain
omega-3 fatty acids).
In another preferred embodiment, said edible oil is in the form of
triglycerides,
s diglycerides or monoglycerides or any mixture thereof, preferably of
unsaturated fatty
acids (e.g. of long-chain unsaturated fatty acids) more preferably of
polyunsaturated
fatty acids (e.g. of long-chain polyunsaturated fatty acids) most preferably
of omega-3
fatty acids (e.g. of long-chain omega-3 fatty acids).
In one preferred embodiment, said edible oil is in the form of free fatty
acids (e.g, long-
chain free fatty acids), preferably free unsaturated fatty acids (e.g. long-
chain
unsaturated fatty acids), more preferably free polyunsaturated fatty acids
most
preferably free omega-3 fatty acids (e.g. of long-chain omega-3 fatty acids).
In one embodiment, the edible oil or an oil derived therefrom contains >40 %
ethyl
esters by weight of the edible oil, >50 % ethyl esters by weight of the edible
oil, >60 %
ethyl esters by weight of the edible oil, preferably >70 % ethyl esters by
weight of the
edible oil, even more preferably >80 % ethyl esters by weight of the edible
oil'and most
preferably >90 % ethyl esters by weight of the edible oil, such as e.g. >99 %
or 100%
ethyl esters by weight of the edible oil. Said ethyl esters preferably being
ethyl esters of
omega-3 fatty acids.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains >30 % triglycerides by weight of the edible oil,
preferably
>35 % triglycerides by weight of the edible oil, even more preferably >40 %
triglycerides by weight of the edible oil (e.g. >45 %, >50 % or >55 %) and
most
preferably >60 % triglycerides by weight of the edible oil, such as e.g. >65
%, >70 %,
>75 %, >80 %, >85 %, >90 % or >95 % triglycerides by weight of the edible oil.
Said
triglycerides preferably being triglycerides of omega-3 fatty acids.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains >30 % unsaturated fatty acids by weight of the
edible oil,
preferably >35 % unsaturated fatty acids by weight of the edible oil, even
more
preferably >40 % unsaturated fatty acids by weight of the edible oil (e.g. >45
%, >50 %
or >55 %) and most preferably >60 % unsaturated fatty acids by weight of the
edible
oil, such as e.g. >65 %, >70 %, >75 %, >80 %, >85 %, >90 % or >95 %
unsaturated
fatty acids by weight of the edible oil.
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19
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains >30 % polyunsaturated fatty acids by weight of the
edible
oil, preferably >35 % polyunsaturated fatty acids by weight of the edible oil,
even more
preferably >40 % polyunsaturated fatty acids by weight of the edible oil (e.g.
>45 %,
>50 % or >55 %) and most preferably >60 % polyunsaturated fatty acids by
weight of
the edible oil, such as e.g. >65 %, >70 %, >75 %, >80 %, >85 %, >90 % or >95 %
polyunsaturated fatty acids by weight of the edible oil.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains >30 % omega-3 fatty acids by weight of the edible
oil,
preferably >35 % omega-3 fatty acids by weight of the edible oil, even more
preferably
>40 % omega-3 fatty acids by weight of the edible oil (e.g. >45 %, >50 % or
>55 %)
and most preferably >60 % omega-3 fatty acids by weight of the edible oil,
such as e.g.
>65 %, >70 %, >75 %, >80 %, >85 %, >90 % or >95 % omega-3 fatty acids by
weight
of the edible oil.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains more than 2 % partial glycerides by weight of the
edible oil,
more preferably at least 3 % partial glycerides by weight of the edible oil,
even more
preferably at least 5 % partial glycerides by weight of the edible oil and
most preferably
at least 7 % partial glycerides by weight of the edible oil, such as e.g. at
least 8, 10, 12,
15, 17, 19, 21, 23, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 % partial
glycerides by weight
of the edible oil.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains more than 2 % diglycerides by weight of the edible
oil, more
preferably at least 3 % diglycerides by weight of the edible oil, even more
preferably at
least 5 % diglycerides by weight of the edible oil and most preferably at
least 7 %
diglycerides by weight of the edible oil, such as e.g. at least 8, 10, 12, 15,
17, 19, 21, 23,
25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 % diglycerides by weight of the
edible oil.
In another embodiment according to the present invention, the edible oil or an
oil
derived therefrom contains more than 2 % monoglycerides by weight of the
edible oil,
more preferably at least 3 % monoglycerides by weight of the edible oil, even
more
preferably at least 5 % monoglycerides by weight of the edible oil and most
preferably
at least 7 % monoglycerides by weight of the edible oil, such as e.g. at least
8,.10, 12,
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15, 17, 19, 21, 23, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 % monoglycerides
by weight
of the edible oil.
In one embodiment according to the present invention, said edible oil or an
oil derived
s therefrom contains less than 70 % partial glycerides by weight of the edible
oil, more
preferably less than 60 % partial glycerides by weight of the edible oil, even
more
preferably less than 50 % partial glycerides by weight of the edible oil and
most
preferably less than 40 % partial glycerides by weight of the edible oil, such
as e.g..less
than 30, 20, 10, 8, 6, 4 or 3 % partial glycerides by weight of the edible oil
(e.g. 2 %.or
io less than 2 % partial glycerides by weight of the edible oil).
In one embodiment according to the present invention, said edible oil or an
oil derived
therefrom contains less than 70 % diglycerides by weight of the edible oil,
more
preferably less than 60 % diglycerides by weight of the edible oil, even more
preferably
is less than 50 % diglycerides by weight of the edible oil and most preferably
less than 40
diglycerides by weight of the edible oil, such as e.g. less than 30, 20, 10,
8, 6, 4or 3
diglycerides by weight of the edible oil (e.g. 2 % or less than 2 %
diglycerides by
weight of the edible oil).
20 In one embodiment according to the present invention, said edible oil or an
oil derived
therefrom contains less than 70 % monoglycerides by weight of the edible oil,
more
preferably less than 60 % monoglycerides by weight of the edible oil, even
more
preferably less than 50 % monoglycerides by weight of the edible oil and most
preferably less than 40 % monoglycerides by weight of the edible oil, such as
e.g. less
than 30, 20, 10, 8, 6, 4 or 3 % monoglycerides by weight of the edible oil
(e.g. 2 %'or
less than 2 % monoglycerides by weight of the edible oil).
By the expression "marine oil" there is meant a marine oil or an oil derived
therefrom.
Examples of oils derived from an edible oil are ethyl esters, free fatty acids
and oniega-
3 fatty acid concentrates. Preferably, said marine oil is an edible marine
oil, such as fish
oil; and an oil derived therefrom is preferably ethyl esters (preferably ethyl
esters of
omega-3 fatty acids such as e.g. ethyl ester concentrates of omega-3 fatty
acids), free
fatty acids or omega-3 fatty acid concentrates produced from a marine oil such
as e.g.
fish oil.
The composition according to the present invention comprises green tea extract
or an
active fraction thereof. By the expression "green tea extract or an active
fraction
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21
thereof' there is meant any extracts obtainable from green tea which have
antioxidant
activity. Antioxidant activity might be measured by a number of methods such
as the
Oxipres method disclosed in example 2.
s The major tea catechins are epigallocatechin gallate (EGCG),
epigallocatechin (EGC),
epicatechin gallate (ECG), and epicatechin (EC). Of these, EGCG is the most
abundant
and possesses the most potent antioxidative activity.
Thus, it is preferred that said green tea extract, or an active fraction
thereof, comprises
at least one catechin, more preferably the green tea extract comprises
epigallocatechin
gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) or
epicatechin (EC)
or any mixture thereof, and even more preferably it comprises epigallocatechin
gallate
(EGCG).
It is preferred that the total amount of catechin(s) in said green tea
extract, or an active
fraction thereof, is as high as possible, e.g. in the range 1-80 %, in the
range 1.0-70 %, in
the range 20-50 % or in the range 20-30 % by weight of the extract.
Further, it is preferred that said green tea extract is added to a carrier. As
shown in
example 3, a composition prepared using green tea extract on a carrier was
found
significantly more stable than a composition prepared using green tea extract
without a
carrier. Further, the carrier alone was found not to affect the stability of
the
composition. These results indicate that more of the active components in
green tea
extract may be dissolved in oil containing compositions by using green tea
extract on. a
carrier.
In one preferred embodiment said carrier is a polysaccharide, even more
preferably~said
carrier is maltodextrin. In another embodiment, said carrier is an inorganic
salt such'as
sodium chloride.
It is preferred that the composition of the present invention is subjected to
some kind of
treatment, e.g. filtration, in order to separate said carrier or at least the
undissolved
fraction of the carrier, from the composition. Accordingly, it is preferred
that the
composition of the present invention is free of carrier, at least free of any
undissolved
carrier.
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22
The amount of said carrier is preferably in the range 50-95 %, more preferably
in the
range 60-90 %, even more preferably in the range 70-90 %, most preferably
about 80 %
by weight of the extract. Such an extract is commercially available under the
trade
name GUARDIANTM Green Tea Extract 20M, from Danisco. An extract of this type
can be used to prepare compositions according to the present invention in an
amount of
from 0.001 to 5 % by weight of the composition, preferably in the range 0.002
to 1 %,
more preferably in the range 0.1 to 0.8 % such as in the range 0.44 to 0.64 %.
In one embodiment, at least additional 0.1 mg potassium hydroxide compared to
the
io composition without green tea extract is required to neutralise 1 gram of
the
composition, more preferably at least additional 0.15 mg potassium hydroxide
compared to the composition without green tea extract is required to
neutralise 1 gram
of the composition, even more preferably at least additional 0.2 mg potassium
hydroxide compared to the composition without green tea extract is required to
neutralise 1 gram of the composition and most preferably at least additional
0,25 mg
potassium hydroxide compared to the composition without green tea extract is
required
to neutralise 1 gram of the composition.
In one embodiment, the composition according to the present invention
comprises at
least 0.05 mg catechins per gram composition (such as e.g. at least 0.06,
0.07, 0.08,
0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 or 0.45 mg catechins per gram
composition),
more preferably at least 0.5 mg catechins per gram composition (such as e.g.
at least
0.55, 0.6, 0.65, 0.7, 0,75, 0.8, 0.85, 0.9 or 0.95 mg catechins per gram
composition),
even more preferably at least 1 mg catechins per gram composition (such as
e.g. at least
1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4 or 1,45 mg catechins per gram
composition),
most preferably at least 1.5 mg catechins per gram composition (such as e.g.
at least 1.6,
1.8, 2, 2.2, 2.4, 2.6, 2.8, 3 or 4 mg catechins per gram composition). Said mg
catechins
per gram composition being analysed and calculated as set forth in example 7.
Further, the inventors of the present invention surprisingly discovered that
if the green
tea extract, or an active fraction thereof, is subjected to an extraction
step, e.g. by using
a lower alcohol (C1_6 alcohol) such as e.g. ethanol; a filtration step; and
that component
A is added either to the filtered green tea extract or to the edible oil, the
oil appeared
totally clear with no visible particles and showed a significantly increased
stability.,
(example 9).
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Thus, a fourth aspect of the present invention relates to a method for
manufacturing the
composition according to the present invention, the method comprising the
following
steps:
a) green tea extract or an active fraction thereof is mixed with a lower
alcohol (C1=6
s alcohol), such as ethanol (e.g. abs. ethanol);
b) the product obtained in step a) is subjected to filtration, e.g. filtered
on a nutch;
c) optionally, the filtrate obtained in step b) is mixed with ascorbyl
palmitate;
d) component A, as defined in the third aspect of the present invention, or
partial
glycerides (monoglycerides and/or diglycerides, preferably monoglycerides),
as,',
defined in the first aspect of the present invention, is added to the product
obtained
in step b) or step c); or optionally directly to the edible oil defined in
step g);
e) optionally, at least one tocopherol or tocopherol derivative and/or
rosemary extract
or an active fraction thereof is added to the product obtained in step a), b),
c) or d);
and
f) the lower alcohol is removed;
g) the product obtained in step f) is mixed with an edible oil.
In one preferred embodiment, the filter cake obtained in step b) is washed
with a lower
alcohol (C1_6 alcohol), such as ethanol (e.g. abs. ethanol), and the washing
solution
combined with the filtrate obtained in step b).
In one preferred embodiment, ascorbyl palmitate is added to the filtrate
obtained iii step
b) and the resulting solution shaken until all ascorbyl palmitate is
dissolved.
Preferably, component A is added to the product obtained in step b) or step c)
and the
resulting mixture shaken vigorously to disperse component A.
The lower alcohol is preferably removed, e.g. at a in vacuo at a suitable
temperature, or
by any other method suitable to remove said lower alcohol.
In one preferred embodiment, the product obtained in step e) is mixed with
rosemary
extract (Rosemary Extract Liquide Refined, Vitablend) and tocopherol
(Tocoblend L70
IP, Vitablend). The resulting mixture is then preferably homogenized, e.g. in
vacuo at
60 C on the rotary evaporator, until a viscous, transparent, deep red liquid
is obtained.
As an alternative to the above disclosed method, the inventors of the present
invention
surprisingly also discovered that if a mixture of green tea extract and an
edible oil is
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24
filtered using a filter having a pore size in the range 15-40 m, the oil
appeared totally
clear with no visible particles and showed a significantly increased stability
(example 1
and example 2).
s Thus, a second aspect of the present invention relates to a method for
manufacturing the
composition according to the present invention, the method comprising the
following,
steps:
a) optionally, green tea extract or an active fraction thereof is mixed with
ascorbyl.s
palmitate;
b) an edible oil is mixed with partial glycerides so that the final
composition contains
more than 2 % partial glycerides (monoglycerides and/or diglycerides,
preferably
monoglycerides) by weight of the final composition; or an edible oil is mixed
with
component A, as defined in the third aspect of the present invention;
c) green tea extract or an active fraction thereof or the product obtained in
step a) is
is mixed with the product obtained in step b);
d) the product obtained in step c) is subjected to filtration;
e) optionally, at least one tocopherol or tocopherol derivative and/or
rosemary extract
or an active fraction thereof is added to the product obtained in step a), b),
c) and/or
d).
Now being able to dissolve extracts from green tea in an edible oil, the
inventors of the
present invention have surprisingly found a synergistic antioxidative effect
from
combining a mixture of tocopherols, rosemary extract and ascorbyl palmitate
with, green
tea extract.
Thus, it is preferred that the composition according to the present invention
further,
comprises at least one tocopherol and/or tocopherol derivative; and/or
rosemary extract
or an active fraction thereof; and/or ascorbic acid and/or an ascorbic acid
derivative.
By the expression "tocopherol" there is meant not only alpha-tocopherol but
also beta-,
gamma- or delta-tocopherol as well as any mixture thereof. 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid is also meant to be included in said
group of
tocopherol compounds.
The amount of tocopherol used in the composition will vary depending upon the
potency of the chosen substance or mixture of substances, but will generally
be in the
range of from about 0.01-20% by weight of the composition, preferably in the
range
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0.01-5%, more preferably in the range 0.01-1%, even more preferably in the
range 0.01-
0,1 %, most preferably about 0.1 %. In one embodiment, there is more than one
tocopherol in the mixture; particularly preferred is a mixture containing a-
tocopherol, 1 -
tocopherol, 7-tocopherol and 6-tocopherol. One example of such a mixture is
s Tocoblend L 70 IP from Vitablend. In another embodiment, y-tocopherol is the
only
tocopherol present in the composition.
By the expression "rosemary extract or an active fraction thereof' there is
meant any
extracts obtainable from rosemary which have antioxidant activity. Antioxidant
activity
io might be measured by a number of methods such as the Oxipres method
disclosed;in
example 2.
It is preferred that said rosemary extract is an oil-soluble extract. Such
rosemary
extracts are commercially available from a variety of manufacturers. The
preferred
15 antioxidant fraction of the extract are primarily in the dehydroabeitic
acid class of
diterpenes. Among the specifically identified active ingredients of the
extract are
carnosol, carnosic acid and rosmanol. Thus, in one embodiment said extract, or
an
active fraction thereof, comprises carnosol, carnosic acid or rosmanol or any
mixture
thereof. However, there are other unidentified components of the extract which
also
20 possess antioxidant activity, and these may also be used in the
composition.
One of the preferred rosemary extract is one which contains from about 1-5 %
(w/w)
carnosic acid, from about 2-7 %(w/w) carnosol, and from about 0.11-0.5 %(w/w)
rosmanol ; such an extract is commercially available under the tradename
StabexE;
25 from SKW Chemicals. An extract of this type can be used in an amount of
from about
0.0001 to about 1%, preferably about 0.1-0.5% by weight of the composition.
Another
preferred rosemary extract is an extract which is commercially available under
the
tradename Oleoresin Rosemary Herbalox Brand, from Kalsec. Analysis of one
batch
of this extract showed it to contain approx. 4.1 % carnosic acid, 0.61 %
carnosol and
0.034 % rosmarinic acid. An extract of this type can be used in an amount of
from about
0.0001 to about 1%, preferably about 0.1-0.5%, more preferably about 0.2-0.4%,
e.g
about 0.3% by weight of the composition. Alternatively one or more of the
compounds
carnosic acid, carnosol and/or rosmarinic acid may be added, preferably in a
combined
concentration from about 0.00005 to about 0.05%, preferably about 0.005-0,03%,
more
preferably about 0.01-0.02 by weight of the composition. Carnosic acid is the
preferred
compound to be added.
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26
By the expression "ascorbic acid derivative" there is meant any derivative of
ascorbic
acid such as e.g. ascorbyl palmitate and ascorbyl stearate. Preferably said
ascorbic acid
and/or ascorbic acid derivative is ascorbyl palmitate. One example of a
commercially
available ascorbyl palmitate product is Grindox ascorbyl palmitate from
Danisco.
Preferably, said composition comprises ascorbic acid and/or an ascorbic acid
derivative
in the range of 0,02-0,12 % by weight of the the composition, preferably in
the range
0,04-0,12 % by weight of the the composition, more preferably in the range
0,06-0,10
%, even more preferably in the range 0,07-0,08 %.
Further, it has previously been disclosed that ascorbyl palmitate is very
effective in
slowing down oxidation of lipids in marine oils. However, in order to dissolve
this
compound in oil formulations it is common to add lecithin, a compound which
producers and distributors prefer to avoid since it has to be declared as a
potential
allergen.
Surprisingly, it has now been found that if said ascorbyl palmitate,
preferably ground or
milled ascorbyl palmitate, is premixed with a small amount of oil, preferably
an
oxidative stable oil (e.g. olive oil), the product is easily dissolvable in a
marine oil..
By the expression "small amount of oil" there is meant that the volume of said
oil is less
than 1 % (w/w) of the edible oil according to the present invention,
preferably less than
0.1 % (w/w), e.g. less than 0.01 % (w/w).
Preferably, said ascorbic acid and/or a derivative thereof, the at least one
tocopherol
and/or tocopherol derivative, rosemary extract or an active fraction thereof
is mixed
with said green tea extract before filtering.
Further, it is preferred that both the ascorbic acid and/or a derivative
thereof, the at least
one tocopherol and/or tocopherol derivative, the rosemary extract or an active
fr action
thereof are dissolved in a small amount of oil. Said oil is preferably an
oxidative stabile
oil, such as a vegetable oil, e.g. olive oil such as food grade olive oil. To
facilitate::this
process the oily mixture may be stirred, shaken and/or treated in an
ultrasonic bath at
temperature in the range 10-180 C (such as 160 C), e.g. in the range 20-120 C
(such as
20 C), 50-100 C or 80-90 C (such as 80 C).
Said ascorbic acid and/or a derivative thereof is preferably milled or ground
before it is
mixed with said small amount of an oil. Further, it is preferred that the
milling or
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27
grounding is as gentle as possible, e.g. by a teflon coated magnet stirrer or
a similar-,-
device suitable for large scale production.
Furthermore, it is preferred that said green tea extract is milled or ground.
In one preferred embodiment according to the present invention, the mixture
that is to
be filtered is stirred, shaken and/or treated in an ultrasonic bath at
temperature in the
range 10-180 C (such as 160 C), e.g. in the range 20-120 C (such as 20 C),
50-100 C
or 80-90 C (such as 80 C) before said mixture is filtered through a filter
having a-pore
io size in the range 0.1-100 m, preferably in the range 5-80 m, more
preferably in the
range 10-50 m and most preferably in the range 10-40 m, such as 15-40 gm.
Thus, a further aspect of the present invention relates to a composition
obtainable by the
above mentioned methods.
There are a number of commercial antioxidant formulations suitable for
improving
oxidative stability of a marine oil, one of them being Tocoblend ATR. The
latter,
produced by the company Vitablend; is a commercial liquid antioxidant
formulation
containing the following ingredients: ascorbyl palmitate, natural mixed
tocopherols,
natural rosemary extract, polysorbate 80 and monopropylene glycol.
In order to compare Tocoblend ATR with the composition of the present
invention, four
formulation were made with the same batch of an omega-3-acid triglyceride
concentrate
(EPAX 6000TG, batch no. 2080630) complying with the European Pharmacopoeia
monograph 1352, Omega-3-acid triglycerides (example 5). While the preparation
based
on the composition according to the present invention (preparation B) appeared
as a
suspension of finely divided particles before filtration, the preparation
based on
Tocoblend ATR (preparation D) formed sticky lumps of solids which did not
disperse in
the oil upon heating.
It is assumed that polysorbate 80 and/or monopropylene glycol in some manner
interfere with the surface of the maltodextrin particles present in the green
tea extract
(GUARDIANTM Green Tea Extract 20M, Danisco), resulting in some form of
agglomeration which reduces the solubilisation of the green tea extract.
Surprisingly, in
Preparation B (examples 5) no such agglomeration occur, giving a product that
is far
better stabilised against oxidation.
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In order to investigate this further, several commercially available
emulsifiers and/or
antioxidant formulations containing such emulsifiers were tested as additives
in the
preparation of compositions according to the present invention (example 6).
s Surprisingly, these experiments indicate that the present invention has best
effect when
avoiding the common food additives like lecithin and polysorbate
80/monopropylene
glycol. Contrary to expectations, a formulation with polysorbate 80/propylene
glycol
reduced the solubilisation of green tea extract. Other emulsifying agents,
including;the
commonly used lechitin, increased problems with precipitation during storage
of oil
samples. On the other hand, the citric acid emulsifier gave excellent results
in
combination with a formulation according to the present invention (example 8
and 10).
Accordingly, it is preferred that the composition according to the present
invention is
essentially free of lechitin and/or polysorbate 80 and/or monopropylene
glycol, more
preferably the composition is totally free of lechitin and/or polysorbate 80
and/or
monopropylene glycol. It is also preferred that said composition does not
contain any
synthetic emulsifiers.
Thus, in one preferred embodiment, the composition of the present invention
further
comprises citric acid esters of mono- and diglycerides. The skilled person
will realise
that other food additives/emulsifiers, such as e.g. tartaric acid esters of
mono- and
diglycerides and acetyltartaric acid esters of mono- and diglycerides, also
may have
similar effects as the citric acid based emulsifier.
A fifth aspect of the present invention relates to a nutritional, food or
pharmaceutical
composition comprising the composition according to the first or third aspect
of the
present invention or the composition obtained by the method according to the
second or
fourth aspect of the present invention.
The invention will now be described further with reference to the following
non-
limiting examples.
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EXAMPLES
Example I
Preparation of transparent oil samples having improved oxidative stability
Preparation 1 (ascorbyl palmitate, tocopherols and rosemary extract)
Ascorbyl palmitate (Grindox, Danisco) was milled to obtain a finely divided
powder
and mixed with tocopherol (Tocoblend L 70 IP, Vitablend), rosemary extract
(Herbalox Brand, type 0, NS, Kalsec) and olive oil (Ybarra Virgin) by
stirring at
50 C for 1/2 hour. The weight ratio of the ingredients was ascorbyl palmitate
(13,3 %),
tocopherol (19,1 %), rosemary extract (53,2 %) and olive oil (14,4 %). 5,4
mg/g (mg per
gram marine oil) of the combined ingredients were added to a marine oil (EPAX
6000
TG EPAX) by stirring at 80 C for 20 minutes.
Preparation 2 green tea extract)
5.4 mg/g (mg per gram marine oil) of a green tea extract (GUARDIANTM Green Tea
Extract 20M, Danisco) was stirred in a marine oil (EPAX 6000TG, EPAX) under
heating at 80 C, and filtered while hot through a filter having a pore size in
the range
15-40 m.
Pr aration 3 (cold filtration)
5,4 mg/g of the combined ingredients as described in Preparation 1 and 5,4
mg/g of
green tea extract were mixed with the same marine oil by stirring at 80 C for
20
minutes. The preparation was cooled to 20 C and filtered through a filter
having a pore
size in the range 15-40 pm.
Preparation 4 (hot filtration)
5,4 mg/g of the combined ingredients as described in Preparation 1 and 5,4
mg/g of
green tea extract were mixed with the same marine oil by stirring at 80 C for
20
minutes. The preparation was filtered at 80 C through a filter having a pore
size in the
range 15-40 pm.
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Example 2
Oil samples having improved oxidative stability (Oxipres method)
The stability of the oils prepared in example 1 were tested by logging the
pressure as a
5 function of residence time at 50 C and an oxygen pressure of about 3.5 bars.
(Oxipres
apparatus produced by Microlab, Aarhus, Denmark)
All preparations showed significant improved stability compared to the pure
oil without
any antioxidants. The induction time for Preparation 1 and Preparation 2 was
115 and
10 124 hours, respectively (figure 1). Preparation 3 (278 hours) showed less
stability than
Preparation 4 (302 hours) (figure 1).
Thus, the inventors of the present invention have found a synergistic
antioxidative effect
from combining a mixture of tocopherols, rosemary extract and ascorbyl
palmitate with
is green tea extract.
Example 3
Gree tea extract with or without a carrier
20 In Preparation 2 (example 1), a commercially available green tea extract
which contains
maltodextrin was used. In order to check whether maltodextrin is of importance
or not,
an analysis similar to what was done in example 2 was conducted using a
commercially
available green tea extract that does not contain a carrier (IndenaTM Green
Tea Extract).
For comparison, the preparations were adjusted to contain the same amount of
active
25 ingredients. Even though the composition containing GUARDIANTM Green Tea
Extract
20M was shown to be more stable than the composition containing IndenaTm Green
-Tea
Extract, both products where demonstrated to be significantly more stable than
the
product which has not been mixed with green tea extract (data not shown).
Further,
maltodextrin alone does not affect the stability of the marine oil (Epax
6000TG oil,
30 EPAX).
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Example 4
Oil samples having improved oxidative stability (weight increase experiments)
Preparations similar to those described in Example 1 were made by using
another
s marine oil, EPAX 6000 EE.
Samples (3.0 gram) are stored in Petri dishes (diameter 6 cm) and incubated in
a
cabinet at 30 C, ambient relative humidity. The increase in weight (%), as a
result of
oxidation, is plotted as a function of residence time (figure 2). The time
span from start
of the experiment to the point where the curve rises steeply, is defined as
the induction
time.
As can be seen (figure 2), the three preparations containing antioxidants are
much more
stable than the pure fish oil. However, the two preparations containing green
tea extract
is far more stable than the sample without green tea extract. In accordance
with
example 2, the inventors of the present invention have found a synergistic
antioxidative
effect from combining a mixture of tocopherols, rosemary extract and ascorbyl
palmitate with green tea extract.
Example 5
Comparison with a commercial antioxidant formulation (Oxipres method)
Preparation A (ascorbyl palmitate, tocopherols and rosemary extract)
Ascorbyl palmitate (Grindox, Danisco) was milled to obtain a finely divided
powder
and mixed with tocopherol (Tocoblend L 70 IP, Vitablend), rosemary extract
(Herbalox Brand, type 0, NS, Kalsec) and olive oil (Ybarra Virgin) by
alternately
magnetic stirring and immersing in a ultrasonic bath at 65 C for 15 minutes.
The weight
ratio of the ingredients was ascorbyl palmitate (13,3 %), tocopherol (19,1 %),
rosemary
extract (53,2 %) and olive oil (14,4 %). 5,4 mg/g (mg per gram marine oil) of
the
combined ingredients were added to a marine oil (EPAX 6000 TG EPAX) by
stirring at
80 C for 20 minutes.
Preparation B (ascorbyl palmitate, tocopherols rosemary extracts and green tea
extract)
Preparation B was prepared identical to Preparation 4 in Example 1.
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Preparation C (Tocoblend ATRI)
5.4 mg/g (mg per gram marine oil)of Tocoblend ATRI was added to a marine oil
(EPAX 6000 TG EPAX).
Preparation D (Tocoblend ATRJ and green tea extract)
Preparation D was identical to preparation C, except that green tea extract
was added in
the same manner and in the same concentration as in Preparation B.
'Tocoblend ATR
Tocoblend ATR, produced by the company Vitablend, is a commercial liquid
antioxidant formulation containing the following ingredients: ascorbyl
palmitate, natural
mixed tocopherols, natural rosemary extract, polysorbate 80 and monopropylene
glycol.
The stability of the oil preparations (A-D) were tested by the Oxipres method
under
Is exactly the same conditions as described in example 2.
The induction time for Preparation A and Preparation B was 117 and 315 hours,
respectively, while the induction time for Preparation C and Preparation D was
74 and
89 hours, respectively.
These results demonstrate that an antioxidant composition according to the
present
invention gives far better protection of omega-3-acid triglycerides than the
commercial
formulation Tocoblend ATR, also when the latter is combined with green tea
dissolved
from a carrier according to the present invention.
Example 6
Effect of emulsifiers
Lecithin
Addition of lecithin (1-a-lecithin, 3-sn-phosphatidylcholine from soy beans,
Fluka)
when making an antioxidant preparation as described in Preparation 1, Example
1
(relative weight of ascorbyl palmitate: lechitin = 1:1) resulted in a product
with a
margarine like appearance. Although this formulation appeared less homogenous,
and
was more complicated to handle than the preparation I of Example 1, the
formulation
with lechitin was added to an omega-3 concentrate complying with the European
Pharmacopoeia monograph 2063, Omega-3-acid ethyl esters 60.
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Then green tea on maltodextrin was added under identical conditions as
described in
Preparation 4, Example 1. When stored in a refrigerator, precipitation of the
oil was
observed after only 6 days, making this preparation unsuitable for practical
use. Similar
precipitation, also after 6 days, was observed when the same oil, but with no
addition of
green tea, was stored in a refrigerator. When stored at room temperature,
precipitate
was formed after 6 days in the oil with green tea, and after 14 days in the
oil without'.
green tea.
Although lecithin is supposed to be a suitable additive in order to dissolve
ascorbyl
palmitate, this Example surprisingly show that a preparation without lecithin
is
preferable for making compositions according to the present invention.
Diacetyl tartaric acid ester
An identical experiment was performed to that described for lecithin above,
except that
instead of lecithin a similar amount of the emulsifier Panodan Visco-LO 2000,
Danisco (diacetyl glycerides of tartaric acid made from sunflower oil) was
utilised. The
antioxidant formulation (including separate addition of green tea on a
maltodextrin
carrier according to the present invention) was added to an omega-3
concentrate
complying with the European Pharmacopoeia monograph 2063, Omega-3-acid ethyl
esters 60. Both when stored at room temperature and in refrigerator a
precipitate was
observed in the oil after only 6 days.
Rosemary extract containing diacetyl tartaric ester
An antioxidant formulation was prepared identical to Preparation 4 in Example
1,
except that the rosemary extract was substituted with Rosemary extract liquid
refined,
containing the emulsifier diacatyl acid ester of mono and diglycerides (E472e)
(Vitablend, article no. 51310). This antioxidant formulation was added to an
omega-3
concentrate complying with the European Pharmacopoeia monograph 2063, Omega-3-
acid ethyl esters 60. A precipitate occurred after storage in refrigerator for
7 days,
indicating that even small amounts of the emulsifier diacetyl tartaric ester
is negative for
the applicability of antioxidant formulations according to the present
invention.
Acetic acid ester of monoglycerides
Two identical experiments were performed to that described for diacetyl
tartaric acid
ester above, except that instead of diacetyl tartaric acid ester similar
amounts of two
commercial acetic acid esters of monoglycerides from hydrogenated palm based
oil'
were used; the two acetic acid esters of monoglycerides had a degree of
acetylation of
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34
0.5 and 0.7 respectively. In both cases precipitate was observed after storage
for 6 days,
both at room temperature and in refrigerator.
Citric acid ester of mono- and diglycerides
An identical experiment to that with acetic acid ester of mono- and
diglycerides from
hydrogenated palm oil were performed, except that instead of acetic acid ester
the
emulsifier Grinsted Citrem 2-IN-I cosher, Danisco was used. The product
consists of
a neutralised citric acid ester of mono-diglycerides. The formulation was
added to two
different batches of omega-3 concentrates complying with the European
Pharmacopoeia
monograph 2063, Omega-3-acid ethyl esters 60. When the experiments were
ended,.the
oils had been stored at room temperatures at 11 weeks without formation of any
visible
precipitate.
Example 7
is Effects of partial glycerides
The antioxidant effect of green tea has primarily been attributed to the
polyphenol
content of the tea leaves, commonly known as tea catechins. Based on the
assumption
that the active components of green tea extract to a large extent are present
as
compounds like phenols, the dissolved amount may be analysed by titration with
potassium hydroxide.
Pr paration EE1
0, 0.5, 1, 2, 4, 5.4, 6, 7 and 8 mg/g (mg per gram marine oil) of a green tea
extract'
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in a marine oil (5.6 %
triglycerides (TG); 3.0 % diglycerides (DG); 3.6 % monoglycerides (MG); 87.8
%.Ethyl
esters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered while
hot through
a filter having a pore size in the range 15-40 m.
Preparation EE2
0, 0.5, 1, 2, 4, 5.4, 6, 7 and 8 mg/g (mg per gram marine oil) of a green tea
extract
(GUARDIANTM Green TEA Extract 20M, Danisco) was stirred in a marine oil (0 %
triglycerides (TG); 0.5 % diglycerides (DG),; 12.0 % monoglycerides (MG); 87.5
%
ethylesters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered
while hot
through a filter having a pore size in the range 15-40 m.
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Preparation TGl
3, 4, 5.4, 6, 7 and 8 mg/g (mg per gram marine oil) of a green tea extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in a marine oil (93.6
%
triglycerides (TG); 4.9 % diglycerides (DG); 0.4 % monoglycerides (MG); 0.5 %
Ethyl
5 esters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered while
hot through
a filter having a pore size in the range 15-40 m.
Preparation TG2
0, 0.5, 1, 2, 3, 4, 5.4, 6, 7 and 8 mg/g (mg per gram marine oil) of a green
tea extract
10 (GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in a marine oil
(63.9 %
triglycerides (TG); 31.6 % diglycerides (DG); 1.9 % monoglycerides (MG); 2.5 %
Ethyl
esters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered while
hot through
a filter having a pore size in the range 15-40 m.
is Preparation Water
0, 0.5, 1, 2, 4, 5.4, 6, 7 and 8 mg/g (mg per gram water) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in water at room
temperature.
20 Preparation EE3
0, 0.5, 1, 2, 4, 5.4, 6, 7 and 8 mg/g (mg per gram marine oil) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in a marine oil (0 %
triglycerides (TG); 0 % diglycerides (DG); 0 % monoglycerides (MG); 100 %
Ethyl
esters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered while
hot through
25 a filter having a pore size in the range 15-40 p.m.
The contents of partial glycerides are analysed by size-exclusion
chromatography
similar to the method described in Ph.Eur. monographs 1352 and 2063.
30 Compared with the other preparations (EE I, TG1 and TG2), EE3 was shown to
have
the lowest amount of dissolved catechines (data not shown).
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Calculation of dissolved catechines
0, 0.5, 1, 2, 4, 5.4, 6, 7 and 8 mg/g (mg per gram water) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in water at room
temperature.
Each of the 9 samples is added potassium hydroxide until the composition is
neutralised
using phenolphthalein as indicator, cf. Ph.Eur. Monograph no. 2.5.1.Acid
value.
The amount of added catechines (5.4 nig GUARDIANTM Green Tea Extract 20M
contains about 1.08 mg catechines) is plottet on the X-axis and the additional
amount of
potassium hydroxide compared to the composition without green tea extract
necessary
to neutralise the composition is plottet on the Y-axis as shown in figure 3.
Dissolution in water gives a straight line that can be utilised as a
calibration curve for
is estimation of the dissolved amount in the omega-3 containing oils.
Example 8
Effects of using citric acid ester of mono- and diglycerides
The antioxidant effect of green tea has primarily been attributed to the
polyphenol
content of the tea leaves, commonly known as tea catechins. Based on the
assumption
that the active components of green tea extract to a large extent are present
as
compounds like phenols, the dissolved amount may be analysed by titration
with.
potassium hydroxide.
0, 1, 2, 4 and 6 weight % Citrem was added to an ethyl ester (EPAX 4020 EE; no
detectable amounts of partial glycerides; about 99 % ethyl esters). The
samples were
split and half of the volumes retained as reference. To the other half were
added
antioxidants according to Example 1, Preparation 1 and 2, i.e. 5,4 mg/g of the
mixture
containing ascorbyl palmitate, tocopherols and rosemary extract, plus 5,4 mg/g
-
GUARDIAN Green Tea Extract 20M, Danisco. All samples were heated to 80 C for
20 minutes and filtered according to Preparation 4 (hot filtration). Acid
numbers were
determined according to Ph.Eur. Monograph 2.5.1.
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Table 1
Composition Acid Value
Marine oil + ascorbyl palmitate + tocopherols + rosemary extract + green 1.1
tea extract
Marine oil + ascorbyl palmitate + tocopherols + rosemary extract + green
tea extract + 1 % Citrem by weight of the marine oil
Marine oil + ascorbyl palmitate + tocopherols + rosemary extract + green 1.9 :
tea extract + 2 % Citrem by weight of the marine oil
Marine oil + ascorbyl palmitate + tocopherols + rosemary extract + green 2.3.,
tea extract + 4 % Citrem by weight of the marine oil
Marine oil + ascorbyl palmitate + tocopherols + rosemary extract + green 2.5
tea extract + 6 % Citrem by weight of the marine oil
Marine oil 1.0
Marine oil + 1 % Citrem by weight of the marine oil 1.1
As can bee seen from table 1, increasing concentration of Citrem results in an
increased
acid value which indicates that higher amounts of tea catechins have been
dissolved in
s the marine oil.
Example 9
Improved method for preparation of transparent oil samples
The preparation of transparent oil samples disclosed in example 1 involves
filtration of
the marine oil containing the specified antioxidants ("large" volumes). It has
now
surprisingly been found that filtration of the final composition is not
required if the
green tea extract is subjected to an extraction step, filtration step (small
volume) and
therafter admixed with Citrem before it is mixed with the marine oil.
is
7,5 gram green tea extract (GUARDIAN Green Tea Extract 20M, Danisco) was
stirred with 50 ml abs. ethanol and the resulting slurry filtered on a nutch.
The filter
cake was washed with a little abs. ethanol, and to the combined filtrates was
added 2,1
gram ascorbyl palmitate (Grindox, Danisco). The filtrate was shaken until all
ascorbyl
palmitate was dissolved. The solution was transferred to a rotavapor flask and
combined
with 27 gram Citrem 2-in-1 (Danisco). The mixture was shaken vigorously to
disperse
the Citrem and ethanol removed in vacua at 60 C by spinning the flask on a
rotary.
evaporator. To the residue was added 9,0 gram rosemary extract (Rosemary
Extract"
Liquide Refined, Vitablend) and 3,0 gram tocopherol (Tocoblend L70 IP,
Vitablend).
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The resulting mixture was homogenized in vacuo at 60 C on the rotary
evaporator until
a viscous, transparent, deep red liquid was obtained, 43,2 gram.
The oil appeared totally clear with no visible particles and showed a
significantly
increased stability (data not shown).
Example 10
Effects of citric acid ester of mono- and diglycerides
The antioxidant effect of green tea has primarily been attributed to the
polyphenol,;"
content of the tea leaves, commonly known as tea catechins. Based on the
assumption
that the active components of green tea extract to a large extent are present
as
compounds like phenols, the dissolved amount may be analysed by titration with
potassium hydroxide.
Preparation 4020 EE
0, 0.5, 1, 2, 4, 6, 8 and 9 mg/g (mg per gram marine oil) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) with or without 1 % citrem by
weight of the marine oil was stirred in a marine oil (EPAX 4020EE; no
detectable
amounts of partial glycerides; about 99 % ethyl esters) under heating at 80 C,
and
filtered while hot through a filter having a pore size in the range 15-40 m.
Preparation EPAX 6000 TG/N
0, 0.5, 1, 2, 4, 6, 8 and 9 mg/g (mg per gram marine oil) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) with or without I % citrem by
weight of the marine oil was stirred in a marine oil (EPAX 6000 TG/N ; about
90 %
triglycerides and about 9 % diglycerides) under heating at 80 C, and filtered
while hot
through a filter having a pore size in the range 15-40 atm.
As shown in figure 4 and 5, the preparations containing 1 % citrem by weight
of the
marine oil have higher amounts of dissolved cathechines compared with
preparations
without citrem. This effect has been demonstrated both in triglyceride oils
(figure 5) and
in ethyl ester oil (figure 4).
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Calculation of dissolved catechines
0, 0.5, 1, 2, 4, 6, 8 and 9 mg/g (mg per gram water) of a green tea extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in water at room
temperature.
Each of the 8 samples is added potassium hydroxide until the composition is
neutralised
using phenolphthalein as indicator, of Ph.Eur. Monograph no. 2.5.1.Acid value.
The amount of added catechines (5.4 mg GUARDIANTM Green Tea Extract 20M
io contains about 1.08 mg catechines) is plottet on the X-axis and the amount
of potassium
hydroxide necessary to neutralise the composition* is plottet on the Y-axis as
shown ''in
figure 4 and 5,
* The amount of potassium hydroxide necessary to neutralise the composition is
calculated as follows:
a) composition without Citrem: the amount of potassium hydroxide
necessary to neutralise the composition - the amount of potassium
hydroxide necessary to neutralise the marine oil;
b) composition with Citrem: the amount of potassium hydroxide
necessary to neutralise the composition - the amount of potassium
hydroxide necessary to neutralise the marine oil + Citrem.
Dissolution in water gives a straight line that can be utilised as a
calibration curve for
estimation of the dissolved amount in the omega-3 containing oils.
Example 11
Effects of monoglycerides
The antioxidant effect of green tea has primarily been attributed to the
polyphenol
content of the tea leaves, commonly known as tea catechins. Based on the
assumption
that the active components of green tea extract to a large extent are present
as
compounds like phenols, the dissolved amount may be analysed by titration with
potassium hydroxide.
Preparation
0, 0.1, 0.6, 0.8, 1 and 1.4 mg/g (mg per gram EPAX 4020EE) of a green tea
extract
(GUARDIANTM Green Tea Extract 20M, Danisco) was stirred in EPAX 4020EE (0 %
CA 02771478 2012-02-17
WO 2011/025382 PCT/N02010/000286
triglycerides (TG); 0 % diglycerides (DG); 0 % monoglycerides (MG); 100 %
Ethyl
esters/Free fatty acids (EE/FFA)) under heating at 80 C, and filtered while
hot through
a filter having a pore size in the range 15-40 m. One set of samples was
added 2.1,%
monoglycerides (said monoglycerides being prepared from EPAX 4020EE) by weight
5 of the oily composition, and the other set of samples was not added any
monoglycerides.
The amount of added catechines (5.4 mg GUARDIANTM Green Tea Extract 20M
contains about 1.08 mg catechines) is plottet on the X-axis and the additional
amount of
io potassium hydroxide compared to the composition without green tea extract
necessary
to neutralise the composition is plottet on the Y-axis as shown in figure 6.
As shown in figure 6, the composition that contained monoglycerides was shown
to
have significantly higher amounts of dissolved catechines as compared to
control.
